Aerosol and Air Quality Research, 20: 1974–1986, 2020 ISSN: 1680-8584 print / 2071-1409 online Publisher: Taiwan Association for Aerosol Research https://doi.org/10.4209/aaqr.2020.05.0192 Toxic Assessment of Heavily Traffic-related Fine Particulate Matter Using an in-vivo Wild-type Caenorhabditis elegans Model Meng-Ching Chung1, Kuo-Lin Huang1*, Japheth L. Avelino2, Lemmuel L. Tayo2, Chih-Chung Lin1,3*, Ming-Hsien Tsai4, Sheng-Lun Lin5,6,7, Wan Nurdiyana Wan Mansor8,9, Ching-Kai Su10, Sen-Ting Huang10 1 Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan 2 School of Chemical, Biological and Materials Engineering and Sciences, Mapúa University, Muralla St., Intramuros, Manila 1002, Philippines 3 Institute of Food Safety Management, College of Agriculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan 4 Department of Child Care, National Pingtung University of Science and Technology, Neipu, Pingtung County 91201, Taiwan 5 Department of Civil Engineering and Geomatics, Cheng Shiu University, Niaosong District, Kaohsiung 83347, Taiwan 6 Center for Environmental Toxin and Emerging-Contaminant Research, Cheng Shiu University, Niaosong District, Kaohsiung 83347, Taiwan 7 Super Micro Mass Research and Technology Center, Cheng Shiu University, Niaosong District, Kaohsiung 83347, Taiwan 8 Faculty of Ocean Engineering Technology & Informatics, Universiti Malaysia Terengganu, 21300, Malaysia 9 Air Quality and Environment Research Group, Universiti Malaysia Terengganu, 21300, K. Nerus, Malaysia 10 Department of Internal Medicine, Kaohsiung Veterans General Hospital Pingtung Branch, Neipu, Pingtung 91245, Taiwan ABSTRACT In association with the mortality rate due to air pollution, vehicular emitted fine particles (PM2.5) are a threat to public health. PM2.5-induced in-vivo studies on environmental microorganisms can be used to assess the adverse impacts of PM2.5 on human health. In the present study, the toxicity of traffic-related-air-pollutant (TRAP) PM2.5 was evaluated in the animal model Caenorhabditis elegans (C. elegans) using different toxicological endpoints such as lethality, survivability (lifespan), behavioral (head thrashing and body bending), and reproduction (brood size). The TRAP PM2.5 sample were collected in Taichung City, Taiwan from Mar 24 to April 15 in 2018. Of these 23 day samples, three samples (Days A, B, and C) were randomly selected. The results showed that no immediate lethality was observed after acute (24 h) exposure of the nematodes. On the other hand, sublethal endpoints of reproduction exhibited statistically significant dose-dependent reduction, although Day A and Day C did not decrease the egg-laying capability of the worms. For the neurological toxicity, it is inferred that the higher the PM2.5 concentrations, the more the adverse effects of neurobehavior (head trashing and body bending) it poses on the C. elegans. The lifespans of nematodes exposed to heavily TRAP PM2.5 were significantly shortened compared with those of untreated ones based on survival rate. The nematodes exposed PM2.5 models not only posed potentially adverse health effects on human but also represented ecotoxic impacts on the ecosystem. In conclusion, heavy concentrations of TRAP PM2.5 significantly and severely disrupted toxicological endpoints of neurology and reproduction to C. elegans. TRAP PM2.5 significantly shortened the lifespan of the nematodes compared with the control. TRAP PM2.5 might more severely influenced the specific toxic endpoints, such as lifespan and neurobehavira, in this in-vivo models compared with the reproductive endpoints. Keywords: PM2.5; Traffic related air pollutant (TRAP); C. elegans; Lifespan; Reproduction; Locomotion. INTRODUCTION * Corresponding author. Tel.: +886-8-7703202 ext. 7092 or 7079; Ambient and indoor air pollution, the most important Fax: +886-8-7740256 environmental risk to health, contributed 7 million deaths E-mail address: [email protected] (K.L. Huang); each year in the past decade (WHO, 2016). The risks that [email protected] (C.C. Lin) entail PM2.5 or fine particulate have threatened the mortality Copyright The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited. Chung et al., Aerosol and Air Quality Research, 20: 1974–1986, 2020 1975 of human lives and the adverse health effects associated with indicator that PM2.5 from traffic sources is a significant PM2.5 made it as a major indicator of air particle pollution by contributing factor to the increase of systemic inflammation the World Health Organization (WHO, 2005). Reports have in humans (Brucker et al., 2013; Zhao et al., 2013; Chao et shown that worldwide exposure to outdoor PM2.5 contributed al., 2018). Additionally, a report indicated strong associations to 4.1 million deaths connected with heart disease and stroke, between TRAP PM2.5 exposure and adverse health effects lung cancer, chronic lung disease, and respiratory infections like atopic diseases and allergic sensitization (Morgenstern (Orru et al., 2011; HEI, 2018; Li et al., 2018; Hayes et al., et al., 2008). 2019). The increasing epidemiological studies indicate that The 1 mm long free-living soil nematode used in the animal cardiopulmonary morbidity due to PM2.5 exposure play a model Caenorhabditis elegans (C. elegans) established by part in the development of diabetes mellitus and adverse Sydney Brenner in 1965 plays a key role in the decomposition birth outcomes (Hu, 2009; Crouse et al., 2012; Kloog et al., and nutrient cycling (Sohlenius, 1980). The C. elegans in-vivo 2012; Chen et al., 2013; Burnett et al., 2014; Zanobetti et system has been used as a model for the assessment of toxic al., 2014). PM2.5 is accountable for a substantially larger effects of air pollutants (Zhao et al., 2014; Sun et al., 2015, number of attributable deaths than other more well-known 2016; Yang et al., 2016; Wu et al., 2017; Wang et al., 2018; life behavioral risk factors such as physical inactivity, Chung et al., 2019; Zhao et al., 2019) because it shows much alcohol use, and high sodium intake, and it is equivalent to advantages over other animal models: production of large the attributable deaths caused by high cholesterol and high number of progeny (100–200 from a single hermaphrodite), body mass index (HEI, 2018). transparency, rapid life cycle (3 days), short lifespan (2–3 PM2.5 has chemical constituents such as sulfates, nitrates, weeks), low cost, and easy laboratory cultivation (Brenner, and ammonium and its large surface area enable it to carry 1974; Nass and Hamza, 2007). Moreover, C. elegans follows various toxic compounds such as polycyclic aromatic the widely accepted ethical principles, known as Three Rs hydrocarbons (PAHs), black carbon, phthalates, and heavy (Reduction of the use of higher animals, Refinement of metals (Yue et al., 2006; Labrada-Delgado et al., 2012; Jiang current techniques, and Replacement of animals with et al., 2019; Wang et al., 2019; Xing et al., 2020). PM2.5 can alternative methods) (Brenner, 1974). be emitted directly into the air due to anthropogenic The toxicity effects of PM2.5 have been successfully activities or it can be formed in the atmosphere, creating examined using the C. elegans animal model. Coal secondary particles (Lu et al., 2019; Lisetskii et al., 2019). combustion-related PM2.5 can induce deficits in the lifespan, PM2.5 has also been identified as one of the major contributor development, reproduction, and locomotion behavior of in traffic-related air pollution (TRAP) (Chao et al., 2018; C. elegans by altering the expression patterns of genes Xiang et al., 2019; Min et al., 2020). Motor vehicle traffic is related to the control of oxidative stress (Sun et al., 2015). an important source of harmful emissions of PM2.5 in the Specifically, more severe toxicity was observed for prolonged cities of developing countries (Kinney et al., 2011; Brown PM2.5 exposures compared to short-term exposures (Sun et al., et al., 2019). According to Kinney et al., (2011), daytime 2015). In addition, Sun et al. (2016) found that heavy metals concentrations of PM2.5 at the sites adjacent to roadways such as lead, chromium, and copper in coal combustion- –3 ranged from 50.7‒128.7 µg m which were higher than related PM2.5 induced lower responses on locomotion behavior WHO’s 24-hour average guideline (25 µg m–3) (WHO, and lifespan of nematodes, and the combined exposure to these 2005). A similar study conducted by Brown et al., (2019) metals caused greater toxicity than single metal exposure. also reported elevated PM2.5 concentrations at the near-road Similarly, Yang et al. (2016) pointed out that certain heavy sampling sites in Denver and Indianapolis. It is indicative metals in ambient PM2.5 possibly caused a decrease in that traffic emissions contribute to PM2.5 levels in the locomotion behavior and production of intestinal reactive environment and may also cause health damage to people oxygen species (ROS) in C. elegans; moreover, the gene exposed to it. Upon inhalation, PM2.5 can readily pass (mtl-1 and mtl-2) encoded metallothioneins, the proteins through the nose filtration and then deposit at the end of the involving in the control of stress response to heavy metals in respiratory tract, consequently damaging